The effects of selenium and the GPx-1 selenoprotein on the phosphorylation of H2AX

Background

Significant data supports the health benefits of selenium although supplementation trials have yielded mixed results. GPx-1, whose levels are responsive to selenium availability, is implicated in cancer etiology by human genetic data. Selenium's ability to alter the phosphorylation of the H2AX, a histone protein that functions in the reduction of DNA damage by recruiting repair proteins to the damage site, following exposure to ionizing radiation and bleomycin was investigated.

Methods

Human cell lines that were either exposed to selenium or were transfected with a GPx-1 expression construct were exposed to ionizing radiation or bleomycin. Phosphorylation of histone H2AX was quantified by flow cytometry and survival by the MTT assay. Phosphorylation of the Chk1 and Chk2 checkpoint proteins was quantified by western blotting.

Results

In colon-derived cells, selenium increases GPx-1 and attenuated H2AX phosphorylation following genotoxic exposures while the viability of these cells was unaffected. MCF-7 cells and transfectants that express high GPx-1 levels were exposed to ionizing radiation and bleomycin, and H2AX phosphorylation and cell viability were assessed. GPx-1 increased H2AX phosphorylation and viability following the induction of DNA damage while enhancing the levels of activated Chk1 and Chk2.

Conclusions

Exposure of mammalian cells to selenium can alter the DNA damage response and do so by mechanisms that are dependent and independent of its effect on GPx-1.

General significance

Selenium and GPx-1 may stimulate the repair of genotoxic DNA damage and this may account for some of the benefits attributed to selenium intake and elevated GPx-1 activity.     

Slow elimination of phosphorylated histone gamma-H2AX from DNA of terminally differentiated mouse heart cells in situ

Phosphorylation of replacement histone H2AX occurs in megabase chromatin domains around double-strand DNA breaks (DSBs) and this modification (called gamma-H2AX) may serve as a useful marker of genome damage and repair in terminally differentiated cells. Here using immunohistochemistry we studied kinetics of gamma-H2AX formation and elimination in the X-irradiated mouse heart and renal epithelial tissues in situ. Unirradiated tissues have 3-5% gamma-H2AX-positive cells and in tissues fixed 1h after X-irradiation gamma-H2AX-positive nuclei are induced in a dose-dependent manner approaching 20-30% after 3 Gy of IR. Analysis of mouse tissues at different times after 3 Gy of IR showed that maximal induction of gamma-H2AX in heart is observed 20 min after IR and then is decreased slowly with about half remaining 23 h later. In renal epithelium maximum of the gamma-H2AX-positive cells is observed 40 min after IR and then decreases to control values in 23 h. This indicates that there are significant variations between non-proliferating mammalian tissues in the initial H2AX phosphorylation rate as well as in the rate of gamma-H2AX elimination after X-irradiation, which should be taken into account in the analysis of radiation responses.     

Histone H2AX participates the DNA damage-induced ATM activation through interaction with NBS1

Phosphorylated histone H2AX (γ-H2AX) functions in the recruitment of DNA damage response proteins to DNA double-strand breaks (DSBs) and facilitates DSB repair. ATM also co-localizes with γ-H2AX at DSB sites following its auto-phosphorylation. However, it is unclear whether γ-H2AX has a role in activation of ATM-dependent cell cycle checkpoints. Here, we show that ATM as well as NBS1 is recruited to damaged-chromatin in a γ-H2AX-dependent manner. Foci formation of phosphorylated ATM and ATM-dependent phosphorylation is repressed in H2AX-knockdown cells. Furthermore, anti-γ-H2AX antibody co-immunoprecipitates an ATM-like protein kinase activity in vitro and recombinant H2AX increases in vitro kinase activity of ATM from un-irradiated cells. Moreover, H2AX-deficient cells exhibited a defect in ATM-dependent cell cycle checkpoints. Taken together, γ-H2AX has important role for effective DSB-dependent activation of ATM-related damage responses via NBS1.     

Tissue-specific DNA-PK-dependent H2AX phosphorylation and gamma-H2AX elimination after X-irradiation in vivo

Histone H2AX rapidly undergoes phosphorylation at Ser139 (γ-H2AX) in response to DNA double-strand breaks. Although ATM kinase and DNA-PK phosphorylate Ser139 of H2AX in culture cells, the regulatory mechanism of γ-H2AX level remains unclear in vivo. Here, we detected the phosphorylation of H2AX and the elimination of γ-H2AX in the mouse skin after X-irradiation. Furthermore, following X-irradiation, the level of γ-H2AX also increased in mice lacking either ATM or DNA-PK. Although the elimination after X-irradiation was detected in the skin of these mutant mice, the elimination in DNA-PK-deficient mice was slower than that in C3H and ATM knockout mice, suggesting that a fraction of γ-H2AX in the skin is eliminated in a DNA-PK-dependent manner. Although the DNA-PK-dependent elimination of γ-H2AX was also detected in the liver, kidney, and spleen, the DNA-PK-dependent phosphorylation of H2AX was detected in the spleen only. These results suggest that the regulatory mechanism of γ-H2AX level is tissue-specific.     

VRK1 chromatin kinase phosphorylates H2AX and is required for foci formation induced by DNA damage

All types of DNA damage cause a local alteration and relaxation of chromatin structure. Sensing and reacting to this initial chromatin alteration is a necessary trigger for any type of DNA damage response (DDR). In this context, chromatin kinases are likely candidates to participate in detection and reaction to a locally altered chromatin as a consequence of DNA damage and, thus, initiate the appropriate cellular response. In this work, we demonstrate that VRK1 is a nucleosomal chromatin kinase and that its depletion causes loss of histones H3 and H4 acetylation, which are required for chromatin relaxation, both in basal conditions and after DNA damage, independently of ATM. Moreover, VRK1 directly and stably interacts with histones H2AX and H3 in basal conditions. In response to DNA damage induced by ionizing radiation, histone H2AX is phosphorylated in Ser139 by VRK1. The phosphorylation of H2AX and the formation of γH2AX foci induced by ionizing radiation (IR), are prevented by VRK1 depletion and are rescued by kinase-active, but not kinase-dead, VRK1. In conclusion, we found that VRK1 is a novel chromatin component that reacts to its alterations and participates very early in DDR, functioning by itself or in cooperation with ATM.     

PP4 is a gamma H2AX phosphatase required for recovery from the DNA damage checkpoint

Phosphorylation of histone H2AX on Ser 139 (γH2AX) is one of the earliest events in the response to DNA double-strand breaks; however, the subsequent removal of γH2AX from chromatin is less understood, despite being a process tightly coordinated with DNA repair. Previous studies in yeast have identified the Pph3 phosphatase (the PP4C orthologue) as important for the dephosphorylation of γH2AX. By contrast, work in human cells attributed this activity to PP2A. Here, we report that PP4 contributes to the dephosphorylation of γH2AX, both at the sites of DNA damage and in undamaged chromatin in human cells, independently of a role in DNA repair. Furthermore, depletion of PP4C results in a prolonged checkpoint arrest, most likely owing to the persistence of mediator of DNA damage checkpoint 1 (MDC1) at the sites of DNA lesions. Taken together, these results indicate that PP4 is an evolutionarily conserved γH2AX phosphatase.     

Werner syndrome protein associates with gamma H2AX in a manner that depends upon Nbs1

The WRN protein is mutated in the chromosomally unstable Werner syndrome (WS) and the Nbs1 protein is mutated in Nijmegen breakage syndrome (NBS). The Nbs1 protein is an integral component of the M/R/N complex. Although WRN is known to interact with this complex in response to gamma-irradiation, the mechanism of action is unclear. Here, we show that WRN co-localizes and associates with gamma H2AX, a marker protein of DNA double strand breaks (DSBs), after cellular exposure to gamma-irradiation. While the DNA damage-inducible Nbs1 foci formation is normal in WS cells, WRN focus formation is defective in NBS cells. Consistent with this, gamma H2AX colocalizes with Nbs1 in WS cells but not with WRN in NBS cells. The defective WRN-gamma H2AX association in NBS cells can be complemented with wild-type Nbs1, but not with an Nbs1 S343A point mutant that lacks an ATM phosphorylation site. WRN associates with H2AX in a manner dependent upon the M/R/N complex. Our results suggest a novel pathway in which Nbs1 may recruit WRN to the site of DNA DSBs in an ATM-dependent manner.     

Critical role of monoubiquitination of histone H2AX protein in histone H2AX phosphorylation and DNA damage response

DNA damage response is an important surveillance mechanism used to maintain the integrity of the human genome in response to genotoxic stress. Histone variant H2AX is a critical sensor that undergoes phosphorylation at serine 139 upon genotoxic stress, which provides a docking site to recruit the mediator of DNA damage checkpoint protein 1 (MDC1) and DNA repair protein complex to sites of DNA breaks for DNA repair. Here, we show that monoubiquitination of H2AX is induced upon DNA double strand breaks and plays a critical role in H2AX Ser-139 phosphorylation (γ-H2AX), in turn facilitating the recruitment of MDC1 to DNA damage foci. Mechanistically, we show that monoubiquitination of H2AX induced by RING finger protein 2 (RNF2) is required for the recruitment of active ataxia telangiectasia mutated to DNA damage foci, thus affecting the formation of γ-H2AX. Importantly, a defect in monoubiquitination of H2AX profoundly enhances ionizing radiation sensitivity. Our study therefore suggests that monoubiquitination of H2AX is an important step for DNA damage response and may have important clinical implications for the treatment of cancers.     

Oxidative stress induces H2AX phosphorylation in human spermatozoa

H2AX phosphorylation occurs following the induction of DNA double strand breaks (DSBs), thus collaborating with many other proteins to mediate important biological functions in somatic cells. In human spermatozoa, the present study showed that H(2)O(2) induced H2AX phosphorylation in a time- and dose-dependent manner. Moreover, such effect could be abolished by the phosphatidylinositol 3-kinase inhibitor wortmannin. Meanwhile, the neutral comet assay also revealed DSBs production in correlation with H2AX phosphorylation assessed by flow cytometry. Besides H2AX phosphorylation, two other collaborating proteins, Rad50 and 53BP1, were also generated in spermatozoa after H(2)O(2) exposure. However, unlike in somatic FL cells, there were no distinctive focuses, but rather a whole nuclei staining pattern of these three proteins in spermatozoa. Additionally, gammaH2AX (the phosphorylated form of H2AX) staining in spermatozoa persisted despite the fact of a decrease in the number of gammaH2AX foci in FL cells after H(2)O(2) removal. Collectively, these results demonstrate that oxidative stress can induce H2AX phosphorylation in human spermatozoa through DSB induction, and that gammaH2AX may be used as a sensitive, novel marker for such DSBs. Moreover, the surveillance system involving gammaH2AX, Rad50, and 53BP1 in human spermatozoa cannot function effectively in DNA repair, but this system may possess other biological functions in response to DSBs.     

AIF promotes chromatinolysis and caspase‐independent programmed necrosis by interacting with histone H2AX

Programmed necrosis induced by DNA alkylating agents, such as MNNG, is a caspase‐independent mode of cell death mediated by apoptosis‐inducing factor (AIF). After poly(ADP‐ribose) polymerase 1, calpain, and Bax activation, AIF moves from the mitochondria to the nucleus where it induces chromatinolysis and cell death. The mechanisms underlying the nuclear action of AIF are, however, largely unknown. We show here that, through its C‐terminal proline‐rich binding domain (PBD, residues 543–559), AIF associates in the nucleus with histone H2AX. This interaction regulates chromatinolysis and programmed necrosis by generating an active DNA‐degrading complex with cyclophilin A (CypA). Deletion or directed mutagenesis in the AIF C‐terminal PBD abolishes AIF/H2AX interaction and AIF‐mediated chromatinolysis. H2AX genetic ablation or CypA downregulation confers resistance to programmed necrosis. AIF fails to induce chromatinolysis in H2AX or CypA‐deficient nuclei. We also establish that H2AX is phosphorylated at Ser139 after MNNG treatment and that this phosphorylation is critical for caspase‐independent programmed necrosis. Overall, our data shed new light in the mechanisms regulating programmed necrosis, elucidate a key nuclear partner of AIF, and uncover an AIF apoptogenic motif.     

Focus on histone variant H2AX: To be or not to be

Phosphorylation of histone variant H2AX at serine 139, named γH2AX, has been widely used as a sensitive marker for DNA double-strand breaks (DSBs). γH2AX is required for the accumulation of many DNA damage response (DDR) proteins at DSBs. Thus it is believed to be the principal signaling protein involved in DDR and to play an important role in DNA repair. However, only mild defects in DNA damage signaling and DNA repair were observed in H2AX-deficient cells and animals. Such findings prompted us and others to explore H2AX-independent mechanisms in DNA damage response. Here, we will review recent advances in our understanding of H2AX-dependent and independent DNA damage signaling and repair pathways in mammalian cells.     

Dynamic change of histone H2AX phosphorylation independent of ATM and DNA-PK in mouse skin in situ

Histone H2AX undergoes phosphorylation on Ser 139 (γ-H2AX) rapidly in response to DNA double-strand breaks induced by exogenous stimuli, such as ionizing radiation. However, the endogenous phosphorylation pattern and modifier of H2AX remain unclear. Here we show that H2AX is regulated physically at the level of phosphorylation at Ser139 during a hair cycle in the mouse skin. In anagen hair follicles, γ-H2AX-positive cells were observed in the outer root sheath (ORS) and hair bulb in a cycling inferior region but not in a permanent superficial region. In telogen hair follicles, γ-H2AX-positive cells were only detected around the germ cell cap. In contrast, following X-irradiation, γ-H2AX was observed in various cell types including the ORS cells in the permanent superficial region. Furthermore, γ-H2AX-positive cells were detected in the skin of mice lacking either ATM or DNA-PK, suggesting that these kinases are not essential for phosphorylation in vivo.     

Autophosphorylation of H2AX in a cell-specific frozen dependent way

Effective cryopreservation is a highly desired outcome in many laboratories including clinical and research centers. Cryopreservation-induced cellular damage through necrosis and apoptosis has been well characterized. However, our knowledge of the mechanism of induction of these cell death modalities is limited. H2AX histone phosphorylation to γ-H2AX is a DNA damage response and is an excellent indicator of DNA double stranded break formation. In this study we examined and detected significant phosphorylation of H2AX in response to cryopreservation of HELF and B16 cell lines. The data provide strong evidence of intrinsic alterations in DNA repair pathway members for which the impact is yet to be fully understood. While further investigation will be necessary to characterize this response, our findings show a clear linkage between freezing resulting in phosphorylation and activation of the key DNA repair enzyme H2AX.     

Post-translational modifications of the N-terminal tail of histone H3 in holocentric chromosomes of Bombyx mori

Epigenetic information is encoded in post-translational modifications (PTMs) of histones. Various combinations of these marks contribute to the regulation of chromatin-templated DNA metabolisms. The histone code is gradually translated into biological responses in model organisms. However, in the silkworm, the modifications of histones with unique holocentric chromosomes have not yet been analyzed. TAU-PAGE analysis of the silkworm histone variants H2A, H2B, and H3, separated by RP-HPLC, suggested silkworm specific modification. Detailed mass spectrometry analyses of the peptides derived from the N-terminus of the silkworm H3.2 generated by glutamyl endopeptidase, lysyl endopeptidase, and trypsin digestions revealed global modifications around H3K9.     

Werner syndrome helicase (WRN), nuclear DNA helicase II (NDH II) and histone gammaH2AX are localized to the centrosome

Werner syndrome helicase (WRN) was found in the centrosome of human cells, both in interphase and in mitosis. Nuclear DNA helicase II (NDH II), also called RNA helicase A (RHA), an interaction partner of WRN, was also present in the centrosome. NDH II localized to the centrosome in interphase but left the centrosome with the ongoing progression of mitosis. The localization of NDH II to the centrosome was hardly affected by cytochalasin D that depolymerizes actin filaments. In contrast, treatment by the microtubules disrupting agent nocodazole strikingly detached NDH II from the centrosome, which was in contrast to WRN that remained there under this condition. Treatment of cells with the DNA damaging agent 4-nitroquinoline-1-oxide (4NQO) released NDH II, but not WRN from the centrosome. Surprisingly, the double-stranded DNA break repair-induced histone variant gammaH2AX was also found in centrosomes of interphase and mitotic cells. Following DNA damage by 4NQO, gammaH2AX left the centrosome with similar kinetics as NDH II. In vitro pull-down assays confirmed a direct physical interaction between these two proteins. Since NDH II associated with gammaH2AX after DNA damage, we suggest that complex formation between NDH II and gammaH2AX may occur in pre-assembled complexes at the centrosome, which are subsequently recruited to sites of damaged DNA for inducing the repair process.     

Solar-simulated light-exposed benzo[a]pyrene induces phosphorylation of histone H2AX

Polycyclic aromatic hydrocarbons (PAHs), wide-spread mutagenic and carcinogenic environmental pollutants, are consistently exposed to sunlight in the environment. The exposure causes structural change, resulting in the generation of a variety of photomodified products having different bioactivities compared with the parent compounds. In this study, we found that benzo[a]pyrene (BaP) exposed to solar-simulated light (SSL)-induced phosphorylation of histone H2AX (γ-H2AX), which was recently identified as an early event after the induction of DNA double strand breaks (DSBs). Although BaP itself did not produce γ-H2AX, SSL-exposed BaP significantly generated γ-H2AX depending on the period of exposure. Furthermore, we revealed that reactive oxygen species produced by the SSL-exposed BaP mainly contributed to the generation of γ-H2AX. The appearance of γ-H2AX means the induction of the most serious form of DNA damage, DSBs, suggesting the potential risk of carcinogenesis.     

γH2AX: A potential DNA damage response biomarker for assessing toxicological risk of tobacco products

Differentiation among American cigarettes relies primarily on the use of proprietary tobacco blends, menthol, tobacco substitutes, paper porosity, paper additives, and filter ventilation. These characteristics substantially alter per cigarette yields of tar and nicotine in standardized protocols promulgated by government agencies. However, due to compensatory alterations in smoking behavior to sustain a preferred nicotine dose (e.g., by increasing puff frequency, inhaling more deeply, smoking more cigarettes per day, or blocking filter ventilation holes), smokers actually inhale similar amounts of tar and nicotine regardless of any cigarette variable, supporting epidemiological evidence that all brands have comparable disease risk. Consequently, it would be advantageous to develop assays that realistically compare cigarette smoke (CS)-induced genotoxicity regardless of differences in cigarette construction or smoking behavior. One significant indicator of potentially carcinogenic DNA damage is double strand breaks (DSBs), which can be monitored by measuring Ser 139 phosphorylation on histone H2AX. Previously we showed that phosphorylation of H2AX (defined as γH2AX) in exposed lung cells is proportional to CS dose. Thus, we proposed that γH2AX may be a viable biomarker for evaluating genotoxic risk of cigarettes in relation to actual nicotine/tar delivery. Here we tested this hypothesis by measuring γH2AX levels in A549 human lung cells exposed to CS from a range of commercial cigarettes using various smoking regimens. Results show that γH2AX induction, a critical event of the mammalian DNA damage response, provides an assessment of CS-induced DNA damage independent of smoking topography or cigarette type. We conclude that γH2AX induction shows promise as a genotoxic bioassay offering specific advantages over the traditional assays for the evaluation of conventional and nonconventional tobacco products.